In Vitro Cell. Dev. Biol.ÐPlant 37:457±461, July±August 2001 DOI:10.

1079/IVP2001201
q 2001 Society for In Vitro Biology
1054-5476/01 $10.0010.00

INDUCTION OF EMBRYOGENIC CALLUS AND CELL SUSPENSION CULTURE FROM SHOOT TIPS
EXCISED FROM FLOWER STALK BUDS OF PHALAENOPSIS (ORCHIDACEAE)

KEN TOKUHARA1* and MASAHIRO MII2

1
Orchid Sanctuary Dogashima, 2848-1 Nishina, Nishiizu-cho, Kamo-gun, Shizuoka 410-3514, Japan
2
Faculty of Horticulture, Chiba University, Matsudo, Chiba 271-0092, Japan

(Received 26 October 2000; accepted 29 March 2001; editor D. D. Songstad)

Summary
Embryogenic calluses were induced from 73% of Phalaenopsis shoot-tip explants excised from flower stalk buds by
culturing for 7 mo. on New Dogashima Medium (NDM) containing 0.5 mM a-naphthaleneacetic acid (NAA), 4.4 mM
6-benzylaminopurine and 29.2 mM sucrose. The sucrose concentration was increased to 58.4 mM 4 mo. after initiation of
the callus culture. These calluses were successfully subcultured as cell suspension cultures in liquid NDM supplemented
with 5.4 mM NAA and 58.4 mM sucrose. By simply reducing the sucrose concentration to 29.2 mM, the cells grew into
plantlets through a developmental process similar to that of Phalaenopsis seedlings. The occurrence of somaclonal variants
was less than 10% in six out of eight genotypes examined. These results suggest that the embryogenic callus and cell
suspension culture could be utilized as the materials for micropropagation and breeding of Phalaenopsis orchids.
Key words: embryogenic callus induction; sucrose concentration; suspension cell culture; shoot-tip culture; flower stalk
buds; Phalaenopsis.

Introduction
Since Morel (1960) reported shoot-tip cultures of Cymbidium,
efficient micropropagation systems using tissue culture techniques
have been reported in many genera of the Orchidaceae (Arditti and
Ernst, 1993). Most of the orchids of commercial importance have
been propagated using tissue culture through the formation of
protocorm-like body (PLB) except for some recalcitrant species
such as Paphiopedilum.
In many plant species, regeneration of plants from callus tissue,
especially highly totipotent embryogenic callus, has been recog-
nized as one of the essential techniques for micropropagation and
biotechnological applications. In orchids, successful reports on
plant regeneration from embryogenic callus have been limited.
Sajise and Sagawa (1991) first reported embryogenic callus
formation in Phalaenopsis without a detailed description of the
method for callus induction. However, there have been no reports
on the induction of embryogenic callus in other orchid species. In
Phalaenopsis, callus induction was achieved from PLB which was
induced by culturing shoot tips of lateral buds on flower stalks
(Ichihashi, 1992) and leaf segments (Ishii et al., 1998). However,
there have been no reports on the direct induction of embryogenic
callus from explants obtained from plants grown in the greenhouse.
In the present study, we show the successful results on the direct
induction of embryogenic callus from shoot-tip explants of flower
*Author to whom correspondence should be addressed: Email tokuhara@
dogashima.com
stalk buds, the establishment of a fine cell suspension, and plant
regeneration through somatic embryogenesis in Phalaenopsis.
Materials and Methods
Callus induction. Greenhouse-grown plants of nine Phalaenopsis
genotypes were used as plant material (Tables 1 and 2). Shoot-tip
explants were obtained from the plants by the method previously
reported (Tokuhara and Mii, 1993) and placed on New Dogashima
Medium (NDM) supplemented with 0.5±10.7 mM a-naphthaleneacetic
acid (NAA), 4.4 mM 6-benzylaminopurine (BA), 29.2 or 58.4 mM
sucrose and solidified with 2 g l21 gellan gum. The pH of the media
was adjusted to 5.4 prior to autoclaving for 15 min at 1208C. Each
explant was inoculated into 15 ml medium in a 24 mm  100 mm test
tube capped with aluminum foil. The cultures were incubated at 23 ^
18C under 14-h photoperiods provided by fluorescent lamps at
33 mmol m22 s21. These cultures were transferred to the same fresh
medium at monthly intervals. After 4 mo. of culture, half of the explants
cultured on medium with 0.5 mM NAA, 4.4 mM BA and 29.2 mM
sucrose were transferred to the same fresh medium with the sucrose
concentration increased to 58.4 mM sucrose. Nine to 11 explants were
cultured for each treatment and the experiment was repeated three
times. After 7 mo. of culture, the rates of callus and PLB formation of
explants were recorded.
Establishment of cell suspension culture. The calluses were induced from
shoot-tips of vegetative buds on the flower stalk of Phalaenopsis [(Baby
HatAnn Jesica)equestris] (PM292) on NDM containing 0.5 mM NAA,
4.4 mM BA, 58.4 mM sucrose and 2 g l21 gellan gum. About 200 mg
calluses were transferred into a 100-ml Erlenmeyer flask containing 40 ml
liquid medium and incubated on a reciprocal shaker at 80 rpm, 23 ^ 18C
under a 14-h light photoperiod provided by fluorescent lamps at
33 mmol m22 s21. After several subcultures at 2-wk intervals, the cell
suspension cultures were used for testing the optimum concentration of plant
growth regulators (PGR). For this study, the cells were collected by
centrifugation at 200  g for 5 min and 1 ml packed cell volume (PCV) (ca.

457
458 TOKUHARA AND MII

TABLE 1

EFFECTS OF NAA AND SUCROSE CONCENTRATION ON CALLUS INDUCTION AND PLB FORMATION FROM SHOOT TIPS EXCISED FROM FLOWER
STALK BUDS OF P.[(BABY HATANN JESSICA)EQUESTRIS] AFTER 7 MO. OF CULTURE

NAA (mM) Sucrose (mM) No. of explants Survival (%) PLB formation (%) Callus formation (%) Fresh weight of callus (mg)
0.5 29.2 27 76.7 ^ 3.2b 44.4 ^ 10.4 24.4 ^ 9.6 73.6 ^ 30.1
0.5 29.2/58.4a 27 78.9 ^ 0.8 5.0 ^ 3.5 73.4 ^ 4.7 200.0 ^ 68.1
0.5 58.4 28 3.7 ^ 3.0 0 0 0
5.4 29.2 32 40.9 ^ 5.7 6.1 ^ 5.0 9.4 ^ 4.3 10.7 ^ 4.7
10.7 29.2 32 31.2 ^ 2.2 3.3 ^ 2.7 16.1 ^ 7.1 14.1 ^ 7.2
Significance * * * *

BA at 4.4 mM was added to all culture media.

a
Sucrose concentration was changed from 29.2 to 58.4 mM after 4 mo. of culture.
b
Means of three replications^standard error.
* Significance at P , 0:05 by ANOVA.

500 mg FW) were transferred into a 100-ml Erlenmeyer flask containing
40 ml of liquid NDM supplemented with NAA (0, 0.5, and 5.4 mM), BA (0,
0.4, and 4.4 mM) and 58.4 mM sucrose. After 4 wk of culture, the cells
(1 ml PCV) in each medium (40 ml) were subcultured in the same fresh
medium and the PCV of these cells was recorded after 4 wk of culture. Five
flasks were made for each treatment.
Plant regeneration and somaclonal variation. After proliferation in
liquid medium, embryogenic cells of eight genotypes (one genotype of P.
Hanaboushi, one genotype of P. Snow Parade, one genotype of P. Little
Steve, three genotypes of P. Wedding Promenade, one genotype of P.
Wedding March and one genotype of P. Reichentea) induced from shoot-tip
culture of vegetative buds on flower stalks, on medium containing 0.5 mM
NAA, 4.4 mM BA, 58.4 mM sucrose and 2 g l21 gellan gum, were
subcultured twice at 1-mo. intervals. For each genotype, 50±100 mg of
the calluses were transferred onto a 9 cm Petri dish containing 25 ml NDM
supplemented with 29.2 mM sucrose and 2 g l21 gellan gum, and cultured
for 5 mo. for PLB formation under the same environmental conditions used
for callus proliferation. Then the PLBs induced were cultured for 10 mo. on
PGR-free medium containing 10 g l21 Potato Granules (Basic American
Foods, Walnut Creek, CA, USA), 10 g l21 apple juice (Kyoei Co. Ltd.
Kuroishi, Aomori, Japan), 58.4 mM sucrose and 2 g l21 gellan gum until
plantlets with four or five leaves and three or four roots were obtained.
Finally, 14±119 plantlets were established in pots for each genotype for
further growth in the greenhouse until flowering. Abnormalities in flower and
leaf characteristics were investigated during the full bloom period in the first
year for each genotype.
Results and Discussion
Callus induction. In the presence of 4.4 mM BA, both the
survival of the explants and callus formation after 4 mo. of culture
were affected by the concentrations of both NAA and sucrose. On
NDM media containing 29.2 mM sucrose, excised shoot-tips turned
pale yellow or necrosed when 5.4 or 10.7 mM NAA was added,
whereas the shoot-tips remained green and induced pale yellow
granular calluses at the base of the explants on a lower
concentration, 0.5 mM, of NAA. PLBs were also induced in several
explants on this medium. However, the use of a higher concentra-
tion, 58.4 mM, of sucrose resulted in complete necrosis of most
explants even at the low NAA concentration after 7 mo. of culture.
Viable explants on this medium induced neither PLB nor callus. In
contrast, the survival rate of the explants on NDM medium
containing 0.5 mM NAA and 4.4 mM BA with 29.2 mM sucrose
after 7 mo. of culture was 77% (Table 1). The change of sucrose
concentration from 29.2 to 58.4 mM, 4 mo. after culture initiation,
had no effect on survival rate. Most of the explants cultured on
29.2 mM sucrose throughout the culture period became green and
44% of the explants induced PLB and 24% formed callus. On the
other hand, explants which were transferred onto medium with

TABLE 2

SOMACLONAL VARIATIONS IN FLOWER, LEAF AND INFLORESCENCE CHARACTERS OBSERVED IN THE PLANTS DERIVED FROM
EMBRYOGENIC CALLUSES USING SHOOT-TIP CULTURE OF VEGETATIVE BUDS ON FLOWER STALK IN PHALAENOPSIS

P. Wedding Promenade
P. Hanaboushi P. Snow Parade P. Little Steve P. Wedding March P. Reichentea
Genotypes PP625 PP1068 PP1674 PP1954 PP1985 PP2352 PP2182 PP2439
No. of regenerants 14 21 41 33 30 36 40 119
No. of variants 2 (14.2)a 0 (0) 0 (0) 2 (6.1) 3 (10) 2 (5.6) 3 (7.5) 57 (47.9)
Creased leaf 2 1
Narrow leaf 2 2 2
Thick leaf and flower 2 5
Labelum-like lateral sepal 1
Dwarfed flower 1 2
Shortened inflorescence axisb 1 1
Shortened internode of inflorescencec 1
Thickened top part of inflorescence axis 46

a
Numbers in parentheses show the percentage for surviving plants.
b
Shortening of the inflorescence axis from base to the node with the first flower.
c
Shorter internodes between each flower.
P. Hanaboushi ˆ P. Carol Ai Killips  P. Otohime; P. Snow Parade ˆ P. Crescent  P. Musashino; P. Little Steve ˆ P. Steven Ai  P. equestris; P.
Wedding Promenade ˆ P. Cosmetic Art  P. equestris; P. Wedding March ˆ P. Hanaboushi  P. equestris; P. Reichentea ˆ P. fasciata  P. gigantea.
 INDUCTION OF EMBRYOGENIC CALLUS 459

Fig. 1. Regeneration of plantlets from embryogenic callus-derived PLB. A, Embryogenic callus derived from the shoot tip of a flower
stalk bud after 7 mo. of culture. B, Cell suspension culture. C, Suspension cells derived from embryogenic callus. D, PLB derived from
suspension cells. E, Elongated PLB with a meristem which is shown as the depression (arrow). F, Root development from PLB at the base
of elongating leaf (arrow). G, Plantlets derived from suspension cells 10 mo. after formation of PLB. Bar ˆ 1 mm; except for C, where
bar ˆ 0.1 mm.

58.4 mM sucrose from that with 29.2 mM sucrose after 4 mo. of
culture became pale yellow and induced callus at 73% instead of
forming PLBs. The calluses induced by this treatment showed the
highest fresh weight (200 mg per explant) after 7 mo. of culture and
were friable (Fig. 1A). In contrast, survival rates of the explants on
media containing high concentrations of NAA at 5.4 and 10.7 mM
were 41 and 31%, respectively. However, most of the surviving
explants only showed slight swelling with browning and the
frequencies of PLB and callus formation were low in these two
media.
There have been several reports on callus formation from the
tissue culture of Phalaenopsis. Sajise and Sagawa (1991) first
reported embryogenic callus formation in Phalaenopsis, but they did
not describe a detailed method for callus induction. Furthermore,
Kobayashi et al. (1993) utilized callus as the source for protoplasts
and first succeeded in plant regeneration from protoplasts in
Phalaenopsis, however, details for callus formation were not
described. Ichihashi (1992) reported that callus-like masses were
induced by culturing shoot tips of young flower stalk buds, in which
excised explants were initially converted into a callus-like mass
followed by development into PLBs. Recently, calluses of
Phalaenopsis were successfully induced on medium supplemented
with 116.9 mM sucrose and 20% coconut water by culturing PLB
segments derived from leaf segments from in vitro-cultured plants
(Ishii et al., 1998). In most of these reports, explants were cultured
without changing the sucrose concentration throughout the whole
culture period. In the present study, the frequency of callus
induction was increased threefold by changing the sucrose
concentration from 29.2 to 58.4 mM 4 mo. after initiation of
culture. In our previous study, white and yellowish PLBs were
induced on media containing 58.4 mM sucrose, and most of the
PLBs died following repeated subculture without inducing callus
on the same medium (Tokuhara and Mii, 1993). A similar
phenomenon has also commonly been observed by commercial
growers in seed germination of Phalaenopsis, in which most of the
green protocorms turned yellow and then died on media contain-
ing 58.4±87.6 mM sucrose. The browning of protocorms could be
reduced by decreasing the sucrose concentration and most of the
protocorms remained green and grew into plantlets on media
containing 29.2 mM sucrose (data not shown). Possibly shoot-tip
explants were similar to protocorms and a high concentration of
sucrose was detrimental to the explants. Once the explants
survived and stabilized their physiological condition at the low
concentration, 29.2 mM, of sucrose during the initial 4 mo. of
culture, they could react to a high sucrose concentration
(58.4 mM) which might be favorable for callus formation. A
high concentration of sucrose might act as an osmotic stress
(George, 1993) or to inhibit chlorophyll formation to induce
embryogenic callus formation.
Establishment of cell suspension culture. The calluses induced
from shoot-tip explants were successfully converted into cell
suspension cultures by transferring into liquid medium on a
reciprocal shaker at 80 rpm (Fig. 1B, C). Proliferation of the cells
was affected by PGR concentrations, although the color of callus
was pale yellow without necrosis in all the culture media tested. The
highest proliferation rate (4.6 times increase in PCV) was obtained
460 TOKUHARA AND MII

Fig. 2. Effects of NAA and BA on proliferation rate of cell suspension

culture of P. [(Baby HatAnn Jessica)equestris]. PCV increase of
suspension cells after 4 wk. PCV was measured by centrifugation at 200 
g for 5 min.

in medium containing 5.4 mM NAA alone for 4 wk, although it is

possible that the optimum concentration may be higher (Fig. 2). The
proliferation rate decreased with increasing BA concentration and
was the lowest (3.1 times increase in PCV) in medium containing
4.4 mM BA alone (Fig. 2).
Plant regeneration and somaclonal variation. Embryogenic
cells of all eight Phalaenopsis genotypes examined could readily
develop into PLBs 5 mo. after transfer onto medium containing a
reduced sucrose concentration, 29.2 mM (Fig. 1D). They grew into
plantlets through a similar developmental process to Phalaenopsis
seedlings when maintained on NDM containing 10 g l21 Potato
Granule, 10 g l21 apple juice, 58.4 mM sucrose and 2 g l21 gellan
gum 10 mo. after PLB formation (Fig. 1E±G). Fourteen to 119
regenerated plants were successfully transferred into pots with
sphagnum moss for each cultivar or genotype. Somaclonal variations
observed in leaf, flower and inflorescence characteristics of some
cultivars were classified into eight categories (Table 2). There were
variants in leaf characteristics such as creased leaf (Fig. 3A),
narrow leaf (Fig. 3B) and thick leaf (Table 3). The variant with thick
leaves and thick flowers was confirmed to be octaploid by flow
Fig. 3. Somaclonal variants derived from embryogenic callus of
cytometric analysis. Two types of flower characteristics were Phalaenopsis. A, Variant with creased leaves (arrow). B, Variant with
observed: labellum-like lateral sepals and dwarfed flowers, both narrow leaves. C, Variant with thickened inflorescence axis at the top part
of which had been observed in our previous study (Tokuhara and (arrow).
Mii, 1998). The remaining three types of variants were those with an
abnormal inflorescence axis. One of the inflorescence variants had
a shorter internode between each flower without any change in
TABLE 3
inflorescence axis length from the base to the node with the first
flower. The second type of variant was characterized by the COMPARISON OF SEVERAL CHARACTERISTICS BETWEEN A
shortening of the inflorescence axis from the base to the node with NORMAL PLANT AND TWO LEAF VARIANTS, ONE WITH NARROW
first flower. These two types of variants were also found in our LEAVES AND THE OTHER WITH THICK LEAVES REGENERATED
previous study (Tokuhara and Mii, 1998). The other type of variant, FROM CALLUS OF P. REICHENTEA (PP2439)
which was found in almost half of the regenerants in PP2439, had a Variant
thicker inflorescence axis from the first flower node to the top than
Characteristic Normal Narrow leaf Thick leaf
normal plants (Fig. 3C). The frequency of somaclonal variants
except for this one was 0±14.2% (Table 2), which was similar to the Length (mm) 92.5 ^ 1.5 65.9 ^ 0.7 78.2 ^ 5.3
values observed in the plants derived from the shoot-tip culture of Width (mm) 44.8 ^ 0.7 24.5 ^ 3.6 40.6 ^ 2.6
Length/width 2.08 ^ 0.03 2.81 ^ 0.38 1.9 ^ 0.07
vegetative buds on flower stalks through the micropropagation of Thickness (mm) 1.8 ^ 0.0 1.9 ^ 0.1 2.6 ^ 0.1
PLBs without callus formation (Tokuhara and Mii, 1998).
In the present study, we established a highly efficient system of On the length of inflorescence axis from base to node with first flower,
embryogenic callus formation from flower stalk buds through shoot- normal plants showed 11:2 ^ 0:4 cm but variant showed 8.2 cm.
 INDUCTION OF EMBRYOGENIC CALLUS
tip culture. The method is expected to be utilized as an efficient
method for micropropagation of Phalaenopsis orchids because of the
high proliferation rate of both the callus and cell suspension culture
without loss of embryogenic potential for at least 5 yr. The
embryogenic callus or cell suspension culture will also be
efficiently utilized as the target material for biotechnological
studies such as somatic hybridizaion and genetic transformation, as
shown in our recent study (Belarmino and Mii, 2000).
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